1. Field of the Invention
The invention is related to the detection of pancreatic lipase. More specifically, the invention relates to feline pancreatic lipase polypeptides, polynucleotides encoding the polypeptides, antibodies specific for the polypeptides, methods for producing recombinant fPLP, methods of using the polypeptides and antibodies to detect pancreatic lipase in biological samples, and methods for diagnosing pancreatitis in an animal.
2. Description of Related Art
Lipases are water-soluble enzymes that hydrolyze water-insoluble substrates into more polar lipolysis products. Several lipases have been identified in microorganisms, plants, and animals (Lin, Y. H., Yu, C., Huang, A. H., 1986. Substrate specificities of lipases from corn and other seeds. Arch. Biochem. Biophys. 244, 346-356; Jaeger, K. E., Ransac, S., Dijkstra, B. W., Colson, C., van Heuvel, M., Misset, O., 1994. Bacterial lipases. FEMS Microbiology Reviews 15, 29-63; Petersen, S. B., Drablos, F., 1994. A sequence analysis of lipases, esterases, and related proteins. In: Woolley, P., Petersen, S. B. (Eds.), Lipases—their structure, biochemistry, and application, Cambridge University Press, Cambridge, pp. 23-48). Lipases share a common triad of amino acids (serine, aspartic or glutamic acid, and histidine) in the active site, which is also shared with serine proteases (Svendsen, A., 1994. Sequence comparisons within the lipase family. In: Woolley, P., Petersen, S. B. (Eds.), Lipases—their structure, biochemistry, and application, Cambridge University Press, Cambridge, pp. 1-21).
Another common feature of almost all lipases is glycosylation site motifs. Many lipases have been shown to be related phylogenetically. The pancreatic lipase gene family is a large gene family with 9 subfamilies (Petersen and Drablos, 1994; Carriere, F., Bezzine, S., Verger, R., 1997. Molecular evolution of the pancreatic lipase and two related enzymes towards different substrate selectivities. Journal of Molecular Catalysis B: Enzymatic 3, 55-64; Carriere, F., Withers-Martinez, C., Van Tilbeurgh, H., Roussel, A., Cambillau, C., Verger, R., 1998. Structural basis of the substrate selectivity of pancreatic lipases and some related proteins. Biochim. Biophys. Acta Rev. Biomembr. 1376, 417-432). In addition there are other groups of phylogenetically related lipases, and yet other lipases that do not belong to a defined gene family (Anderson, R. A., Sando, G. N., 1991. Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases. J. Biol. Chem. 266, 22479-22484).
The main function of lipases is the hydrolysis of lipids. A lipase is needed whenever an apolar lipid needs to cross a biological membrane. Triglycerides are prime examples of apolar lipids. Thus lipase is needed in order for triglycerides to be absorbed from the intestinal tract. There are two digestive lipases in most vertebrate species, i.e., a preduodenal lipase and classical pancreatic lipase (Carriere, F., Gargouri, Y., Moreau, H., Ransac, S., Rogalska, E., Verger, R., 1994. Gastric lipases: cellular, biochemical and kinetic aspects. In: Woolley, P., Peterson, S. B. (Eds.), Lipases—their structure, biochemistry, and application, Cambridge University Press, Cambridge, pp. 181-205). Preduodenal lipase has been shown to originate from a single tissue in all species examined to date. A pharyngeal lipase was identified in cows and sheep, a lingual lipase in rats and mice, and a gastric lipase in human beings, monkeys, horses, pigs, guinea pigs, cats, and dogs. No preduodenal lipase could be identified in chickens. In human beings and dogs it has been shown that gastric lipase contributes significantly to the digestion of dietary triglycerides. However, pancreatic lipase (also called classical pancreatic lipase) is the most important enzyme in the digestion of dietary triglycerides (Carriere, F., Moreau, H., Raphel, V., Laugier, R., Benicourt, C., Junien, J.-L., Verger, R., 1991. Purification and biochemical characterization of dog gastric lipase. Eur. J. Biochem. 202, 75-83; Carriere, F., Barrowman, J. A., Verger, R., Laugier, R., 1993a. Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans. Gastroenterol. 105, 876-888).
It has recently been shown by immunolocalization that pancreatic lipase is detected only in pancreatic acinar cells in clinically healthy animals, suggesting that classical pancreatic lipase may be an ideal marker for function and pathology of the exocrine pancreas (Steiner, J. M., Berridge, B. R., Wojcieszyn, J., Williams, D. A., 2002. Cellular immunolocalization of gastric and pancreatic lipase in various tissues obtained from dogs. Am. J. Vet. Res. 63, 722-727). This hypothesis has been confirmed in clinical studies that have shown that the measurement of pancreatic lipase immunoreactivity in serum is a specific marker for exocrine pancreatic function and also highly sensitive for pancreatitis in the animals, such as dogs (Steiner, J. M., Broussard, J., Mansfield, C. S., Gumminger, S. R., Williams, D. A. 2001a. Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs with spontaneous pancreatitis. J. Vet. Int. Med. 15, 274; Steiner, J. M., Gumminger, S. R., Rutz, G. M., Williams, D. A. 2000b. Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs with exocrine pancreatic insufficiency. J. Vet. Int. Med. 15, 274; Steiner, J. M., Gumminger, S. R., Williams, D. A. 2000 c. Development and validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine pancreatic lipase immunoreactivity (cPLI) in serum. J. Vet. Int. Med. 15, 311).
Pancreatic lipase has an approximate molecular weight of 50 kilodaltons. The purification of classical pancreatic lipase has been reported in many species (Rathelot, J., Julien, R., Bosc-Bierne, I., Gargouri, Y., Canioni, P., Sarda, L., 1981. Horse pancreatic lipase. Interaction with colipase from various species. Biochimie 63, 227-234; Bosc-Bierne, I., Rathelot, J., Perrot, C., Sarda, L., 1984. Studies on chicken pancreatic lipase and colipase. Biochim. Biophys. Acta 794, 65-71; and U.S. Pat. No. 6,855,506).
Pancreatitis is a common condition in cats. Clinical symptoms of pancreatitis are non-specific and the disease can be difficult to diagnose. Thus, most cases remain undiagnosed. Pancreatitis is associated with an increased amount of digestive enzymes and zymogens leaking into the blood stream. One of these enzymes is pancreatic lipase. A number of assays have been developed to detect the presence of lipase in serum by use of catalytic assays. However, these assays lack sensitivity and specificity for pancreatic lipase in both human beings and animals. Lipase levels are affected by both pancreatic and non-pancreatic conditions such as kidney or liver diseases or administration of corticosteroids. Further, many cell types other than pancreatic cells also secret lipases. Thus, the changes in lipase activities do not necessarily reflect the presence of pancreatitis. What is desirable is reagents and a simple and rapid method for sensitive and specific detection of pancreatic lipase protein in a easily obtainable biological sample, without the need of taking biopsy samples and the use of immunohistochemistry.
The Applicants have previously developed an assay for detecting canine pancreatic lipase in a biological sample (co-pending application U.S. patent application Ser. No. 11/107,086, filed Apr. 15, 2005, published as U.S. Patent Application Publication No. 2005-0233368, which is incorporated herein by reference in its entirety). However, because of the innate differences existing between canine and feline, the levels of feline pancreatic lipase in the serum sample of a feline subject with sub-clinical and mild form of pancreatitis are much lower than those in canine. Thus, there exists a need for reagents specific for feline pancreatic lipase protein (fPLP) and a simple and sensitive method to enable accurate detection of fPLP and early diagnosis of feline diseases relating to fPLP.